Electrophysiology and fluorescence microscopy of ligand-gated ion channels

The thesis presented here describes studies of ligand binding, function, and arrangement of ligand-gated ion channels in cell membranes. Fluorescence microscopy and the patch clamp technique are used to investigate the nicotinic acetylcholine receptor and the serotonergic 5-HT3 receptor. Charge effects on ligand binding were investigated on the 5-HT3 receptor by site directed mutagenesis of strongly conserved charged amino acids close to the ligand-binding site. It turns out that steric effects have a higher impact on ligand binding than charges but that charged amino acids change the local environment strongly. This was exploited using a fluorescently labelled ligand to determine the distance of the bound ligand to the mutated amino acid. Ligand binding to the acetylcholine receptor was studied with a novel fluorescent ligand based on a toxin from the venom of marine snails (α-conotoxin GI).The fluorescent toxin acts as antagonist and can be bind completely reversible and with excellent specificity to muscle type acetylcholine receptors. Repetitive cycles of binding and unbinding were performed to obtain binding kinetics on single cells.The developed fluorescence microscope experiment enables the detection of only very low amount of fluorescent ligand from around 103 down to single molecules. The reversible binding was exploited to label single acetylcholine receptor and to monitor their diffusion in cell membranes. HEK293 cells served as a model system where co-expression of the anchoring protein rapsyn strongly influences receptor diffusion. Data were compared to diffusion in muscle cell lines during differentiation into myotubes. To obtain new information on ligand binding and channel gating, an experiment has been developed to simultaneously measure fluorescence and ion channel activity. The fluorescently labelled α-conotoxin GI was used to demonstrate the capability of this system. In addition, to increase the yield of electrophysiological measurements, a technique to perform patch clamp on a chip was further developed towards automation.